This proposal studies how a key innate immune receptor, Nlrp3, contributes to renal tubular epithelial (RTE) cell injury in a murine model of renal ischemia reperfusion (IR) injury. The project is highly significant for clinical obligate ischemic kidney injury (e.g., as in donor kidneys harvested for transplantation). Broad/long-term objectives: The long-term goals of the proposed research are to define how Nlrp3 contributes to injurious tissue responses in the kidney. Specific Aims: The specific objective of this proposal is to test the hypothesis that the cytoplasmic PRR Nlrp3 is a key contributor to RTE cell damage induced by renal IR injury. Aim 1 asks whether Nlrp3 activation directs RTE cell injury, defines the signaling events that lead to this injury, and determines whether Nlrp3 blockade prevents RTE cell injury. Aim 2 asks whether activation of Nrp3 contributes to renal IR injury primarily through direct (local) or indirect (systemic) mechanisms. Research Design and Methods for Achieving the Stated Goals: Aim 1 will test how ligation of Nlrp3 injures RTE cells, whether the Nlrp3 activating stimulus directs the mode of RTE cell injury, how molecules released from necrotic cells (DAMPs) activate Nlrp3-mediated injury of healthy RTE cells, and whether RTE cell injury can be blocked by either blocking Nlrp3 or one of its upstream activating pathways. In vivo IR injury incorporates other mediators of tissue injury, such as inflammation, so aim 2 focus on whether Nlrp3 activation plays a broader role in the kidney by separating local (kidney injury) from systemic (inflammation). Direct (local, kidney specific) effects are separated from indirect (systemic) effects in a kidney transplant model where injury responses of the Nlrp3-/- transplanted kidney are studied in a wild type (WT) host, and WT kidney injury studied in a Nlrp3-/- host. Health Relatedness of Project: If the aims of this proposal are met we will learn how molecules released from injured tissue activate Nlrp3-dependent injurious responses in the kidney. This knowledge is crucial for the development of rational target therapies for prevention or amelioration of renal IR injury in clinical situations where hypoxia is anticipated (e.g., pretreatment of donor kidneys prior to harvest for transplantation). Focusing on the earliest events of ischemic kidney injury holds the greatest promise for effective therapeutic strategies.